Phosphate-crosslinked starch nanoparticles and use for drug delivery

ABSTRACT

This specification describes a nanoparticle delivery agent for drugs such as chemotherapy drugs. Starch nanoparticles are internally crosslinked by a phosphate crosslinker such as sodium trimetaphosphate (STMP) using a phase inversion emulsion process. The particle size may be in the range of 80-500 nm. A wide variety of organic phosphates are present apart from the phosphodiester crosslinking. These included triphosphates, monophosphates and diphosphates. The nanoparticles are hydrogels and retain significant amounts of water when dispersed in solution possibly due to the electrostatic repulsion between the chains within the nanoparticle. The nanoparticles are, in general, non-toxic, for example to HeLa cancer cells. The nanoparticles display a high drug loading, with a maximum seen with about 20-40 mol % STMP. Drug release occurs more readily at lower pH. Exposure to typical cell culture environments induces significant release of drug compared to simple buffer environments.

RELATED APPLICATIONS

This application is a continuation-in-part of International Application No. PCT/US2019/024619, filed Mar. 28, 2019, which claims the benefit of U.S. provisional application 62/648,986 filed on Mar. 28, 2018 US and provisional application 62/661,669 filed on Apr. 24, 2018; and also directly claims the benefit of U.S. provisional application 62/661,669 filed on Apr. 24, 2018. All of the applications mentioned in this paragraph are incorporated by reference.

FIELD

This specification relates to nanoparticles, such as starch nanoparticles crosslinked with a phosphate such as STMP, and to methods of making nanoparticles. The specification also relates to the use of nanoparticles for drug delivery, for example in a method of treating cancer.

BACKGROUND

International Publication Number WO 2013/081720 A1, Aptamer Bioconjugate Drug Delivery Agent, describes starch nanoparticles used as a drug delivery device. The delivery device may be in the form of an aptamer-biopolymer-active agent conjugate wherein the aptamer targets the device for the treatment of cancer. The delivery device survives for a period of time sufficient to allow for transport and uptake of the delivery device into targeted cells. The release profile can be modified by the degree of crosslinking. The nanoparticles may be made by applying a high shear force in the presence of a cross-linker. The nanoparticles may be predominantly in the range of 50-150 nm and form a colloidal dispersion of crosslinked hydrogel particles in water.

INTRODUCTION

The following introduction is intended to introduce the reader to the detailed description to follow and not to limit or define any claimed invention.

This specification describes a nanoparticle and its use as a delivery agent for drugs such as chemotherapy drugs. The nanoparticles are primarily made up of starch polymer molecules that have been crosslinked together by a phosphate crosslinker such as sodium trimetaphosphate (STMP) optionally using a phase inversion emulsion process. The particle size is in the range of 80-500 nm, for example 80-300 nm or 200-500 nm. A wide variety of organic phosphates may be present in the nanoparticles in addition to phosphodiester crosslinking, for example triphosphates, monophosphates and diphosphates. The nanoparticles are hydrogels and swell to retain water when dispersed in an aqueous solution. Without intending to be limited by theory, the swelling may due to electrostatic repulsion between the molecular chains within the nanoparticle. The nanoparticles are, in general, non-toxic, for example to mammalian cells such as HeLa cancer cells. The nanoparticles are capable of a high drug loading, with a maximum seen at about 20-40 mol % STMP, for example with 30 mol % STMP. Drug release occurs more readily at lower pH. Exposure to typical cell culture environments induces significant release of drug compared to simple buffer environments.

In a process described herein, biopolymer-based nanoparticles are made using an emulsion process such as a phase inversion emulsion process. The biopolymer may be starch. The biopolymer is cross-linked with a phosphate cross-linker, for example STMP. Optionally, one or both of a salt and caustic are present in the water phase of a water-in-oil emulsion. The water phase also contains the biopolymer and phosphate cross-linker. The nanoparticles may have a negative zeta potential when dispersed in an aqueous composition such as water or blood at about neutral pH. Optionally, the nanoparticles may have an average particle size of 80 nm or more or 200 nm or more. The nanoparticles may be targeted with a ligand such as an aptamer.

A nanoparticle described herein comprises a biopolymer, phosphorous, an active agent and optionally a targeting agent. The biopolymer may be starch. The starch may make up 50% or more of the mass of the nanoparticle. Optionally, the starch may be 80% or more or all of the polymers present in the nanoparticle. The phosphorous may include one or more starch-phosphate compounds. Optionally, the nanoparticle has a negative zeta potential at a pH of 7.0. Optionally, the nanoparticles may have an average size in the range of 80-500 nm, 80-300 nm or 200-500 nm as determined by the peak intensity size or Z-average in dynamic light scattering (DLS).

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is schematic representation of a process of making a drug delivery vehicle by crosslinking starch chains followed by conjugation with a ligand such as an aptamer.

FIG. 2 is a schematic representation of a process of making an STMP crosslinked starch nanoparticle (SNP) using phase inversion emulsion wherein, initially, an O/W emulsion is formed which, after an increase in temperature, becomes an O/W emulsion and the STMP is added so that the crosslinking reaction occurs within the emulsion droplet.

FIG. 3 is graph of conductivity measurements during phase inversion wherein a clear drop in conductivity is seen starting at 30° C., indicating the phase inversion process had begun; once it levelled off, the process was complete.

FIG. 4 is graphs of dynamic light scattering (A) and ζ-Potential (B) measurements for the various synthetic formulations of STMP-SNPs wherein there is some increase in particle size, and negative surface charge increases with increasing STMP content in the synthesis.

FIG. 5 is TEM Images of STMP-SNPs prepared using 0 (A), 1 (B), 5 (C), 10 (D), 30 (E) and 50 (F) mol % STMP.

FIG. 6 is SEM images of samples prepared without STMP (A) and with 10 mol % STMP (B).

FIG. 7 is P-NMR of samples prepared with 0 (A), 1 (B), 5 (C), 10 (D), 30 (E) and 50 (F) mol % STMP; wherein the overall nature of the species present was the same in each sample, but at higher concentrations of STMP, larger amounts of inorganic species were present.

FIG. 8 shows the mass of water retained in nanogels prepared at different STMP concentrations (A) and iodine stained gels of the centrifuged product (B). In general, there was very little water retention at low STMP concentrations, with a significant increase at 30 mol %.

FIG. 9 shows the mass of water retained by the 30 mol % nanogel as a function of salt concentration (A) and an image of the iodine-stained product with increasing NaCl concentration after centrifugation (B). The increase in salt concentration dramatically decreases the amount of water retained, with Mg²⁺ being more effective than Na⁺.

FIG. 10 is an MTT Assay for 0 mol % and 30 mol % samples with HeLa cells.

FIG. 11 is a calibration curve for DOX found by fluorescence spectroscopy.

FIG. 12 shows drug loading capacity of 30 mol % STMP-SNPs with pH (A) and drug release profiles of the same sample at different pH (B). For the release profile, the drug was loaded at pH 7.6.

FIG. 13 shows drug loading capacity at pH 7.6 for STMP-SNPs prepared at different concentrations of STMP (A) and release profiles of these loaded STMP-SNPs at pH 4 (B).

FIG. 14 shows drug release in various cell culture environments. The release was much more apparent in these mixtures compared to a simple buffer.

DETAILED DESCRIPTION

Along with surgery and radiation treatment, chemotherapy is widely used in the treatment of cancer. This involves using cytotoxic drugs to directly kill the cancer cells. For example, doxorubicin intercalates with DNA and is especially toxic to rapidly dividing cells such as cancer cells. One of the major limiting factors is that these drugs are non-discriminating; they affect both healthy and diseased cells. It is therefore useful to localize chemotherapy to the site of action, sparing healthy cells. The drug must be protected to some degree until it is internalized into the cancer cell for immediate action.

In the context of drug delivery, it is useful to enhance specificity; carrying the drug preferentially to the diseased cells and leaving most healthy cells unaffected. To this end, active targeting can involve another step in the design of the nanocarrier: conjugation with a binding ligand. Most cancer cells have, on their surface, macromolecules (such as proteins) that will trigger internalization of an external agent within their cell walls through some binding event. These macromolecules are known as receptors and the process by which the external agent is internalized is known as receptor-mediated endocytosis. If a nanoparticle is conjugated with a ligand that binds to this receptor, the cell will internalize it. The receptors on the surface of a cancer cell will be present only for that type of cancer, allowing for selective targeting and sparing healthy cells. The ligand could be a protein, small molecule, or even DNA, as long as there is a significant binding event.

There are several aptamers which have been found from cell-SELEX that have demonstrated specificity and high affinity for certain cancer cell lines. Some of these aptamers are summarized in Table 1. These aptamers are classed according to which receptors they bind to, and not according to the cancer cells themselves. This is because many cancer cell lines may have similar receptors on their surface and would internalize the DNA aptamer in the same way.

TABLE 1 Selected aptamers developed by cell-SELEX Aptamer Target AS1411 Nucleolin sgc8c Protein Tyrosine Kinase 7 (PTK7) 5TR1 Mucin 1 (MUC1) A10 Prostate-Specific Membrane Antigen (PSMA) S11e Unknown (specific to A549 lung cancer cells)

Starch (or more specifically, starch nanoparticles) may be used as a drug delivery vehicle since, among other things, it is biocompatible. Starch is a polysaccharide derived from plants and is composed of two different polymers: amylose and amylopectin. For example, starch derived from corn contains 27% amylose and 73% amylopectin, whereas potato starch is composed of 20% amylose and 80% amylopectin.

Native starch is not soluble in water at room temperature. This is because starch exists as granules that are typically several tens of microns in size. The process of starch gelatinization in water is known as cooking and is one of the main reactions performed to make starch soluble. Gelatinization can occur almost spontaneously in alkaline conditions (more specifically, pH>10) without significant heating due to increased rate of hydration of the granules.

A hydroxyl group on starch can be linked to another hydroxyl group from another starch chain with a bifunctional small molecule. This results in the formation of a crosslink between the two chains. Crosslinking smaller starch particles provides more stability and resistance to degradation.

STMP is a biocompatible and non-toxic crosslinker that can be used for starch. It consists of three phosphate groups arranged in a cyclic manner, with alternating phosphorus and oxygen atoms completing a 6-membered ring. It is a FDA-approved thickening agent. At a sufficiently high alkalinity (pK_(a) for hydroxyl groups ˜12.6), the hydroxyl groups on the sugar rings become deprotonated (forming an alcoholate) and the oxygen ion can attack one of the phosphorus atoms on the ring through a nucleophilic reaction mechanism. Another hydroxyl group on starch attacks the same phosphorus atom, forming a phosphate bridge between the two sugar rings.

In reality, the crosslinking reaction is much more complicated, and it is somewhat inefficient. Sang et al. performed extensive P-nuclear magnetic resonance (NMR) studies to determine the extent to which side reactions may dominate the crosslinking reaction. The first step of the reaction is the nucleophilic attack of the starch alcholate on the STMP ring to form monostarch triphosphate. At this point, two different reactions may occur if the pH is maintained between 11.5 and 12.5. The first is with another starch alcholate attacking the same phosphorus forming the desired crosslink (distarch monophosphate). The second reaction that may occur is with a hydroxyl group (supplied by alkaline conditions) attacking the same phosphorus, forming monostarch monophosphate, which is quite stable. Lastly, a peeling reaction can occur at lower pH where a phosphate group from the monostarch triphosphate can migrate off, eventually (in the presence of water) forming the HPO₄ ²⁻ anion. These findings were also supported by Lack et al., who performed similar studies using a model system and arrived at a similar conclusion.

In some examples, only about 50% of the STMP added initially actually reacts with starch to form the triphosphate. Of that amount, about 20% goes on to form the distarch monophosphate (the other roughly 80% being various other phosphate species, including triphosphates and pyrophosphates). Therefore, with respect to the amount of STMP that is added, the reaction itself is relatively inefficient. The addition of salts (such as sodium chloride) increases the efficiency of the reaction. This crosslinking process is also temperature dependant, with higher temperatures resulting in more phosphorus incorporation.

Bulk starch would not be useful for applications like drug delivery since the size of the granules are simply too big. Wth starch, crosslinking of the soluble (i.e. cooked and/or chemically and/or thermo-mechanically degraded) polymer strands can be performed using STMP or sodium tripolyphosphate (STPP). STPP is similar to STMP in that a phosphate linkage is formed between the two starch chains, but the mechanism is slightly different. The crosslinked strands form a nanoparticle, which may be a colloidal hydrogel and can be used in a dispersion or suspension.

When aptamers bind to receptors on the cell surface, the process of internalization begins where cell membrane collapses to engulf the particle in a vesicle, pushing it into the cytoplasm. This internalization is known as receptor-mediated endocytosis. This phenomenon is promoted by the multi-valence effect, where the more aptamer on the surface of the particle (per unit of surface area), the stronger the binding will be and the more internalization will occur. If the weight ratio of DNA to starch is kept constant, there is much higher coverage of DNA on a larger nanoparticle.

Another aspect of particle size lies in the drug loading capacity. Drugs may be loaded into a porous and/or hydrogel starch nanoparticle structure. If each particle is assumed to be spherical, then a small sphere would have a certain drug loading within it. Evidently, this drug loading is limited by the volume and thus would be a function of the radius of the sphere. If the radius of the sphere were to be increased by 10, then the volume and drug loading would increase by 1000-fold. Ignoring the cost of the vehicle itself, the main factor to consider (economically) is how many drug molecules each aptamer may carry. In that regard, the drug/aptamer ratio becomes important. Aptamer coverage on the nanoparticle surface is limited by surface area, while drug loading is limited by volume. Therefore, increasing the particle size by 10-fold would increase the drug/aptamer ratio by 10 fold.

A starch nanoparticle may have a size (i.e. peak intensity or Z-average diameter) in the range of 80-500 nm, 80-300 nm or 200-500 nm, with a high molecular weight (achieved by crosslinking minimally degraded strands of starch, the strands having a molecular weight of 100,000 Da or more) so that more drug molecules could be loaded into porous/hydrogel starch structure, and for more aptamer coverage to exploit the multi-valence effect in cellular uptake. Nanoparticles intended delivered in the blood (either by injection or after oral delivery and uptake) are preferably smaller than 300 nm, for example 80-300 nm since nanoparticles that are too large are easily cleared by the body. Nanoparticles delivered by other means, for example by injection into a tumor or contact with a mucosal membrane or body cavity, are preferably larger than 200 nm, for example 200-500 nm, for further improved drug loading or drug to ligand ratio. Crosslinking with STMP also adds a highly negative charge to the particle to potentially further improve the drug loading capacity. This charge may reduce in acid environments such as a tumor site to enhance drug release at the treatment site.

This specification describes making starch nanoparticles for purposes of drug delivery. Free starch chains are crosslinked by STMP in a controlled manner to form high molecular weight (MW) particles, optionally followed by conjugation with a ligand such as a DNA aptamer for cell-internalization, as shown in FIG. 1.

To confine the particle size to sub-micron regimes, emulsions (more specifically, water in oil (W/O) emulsions) can be used. Since the STMP is hydrophilic, it will partition into the water phase. The maximum size of the particle is determined by the droplet size during the emulsion process as the particles would be internally crosslinked within it.

When one homogenizes a mixture of a water phase and an oil phase in the presence of a surfactant, an emulsion is formed where one phase (the dispersed phase) is suspended in the other phase (the continuous phase) as droplets. The resulting emulsion formed can be oil-in-water (O/W) or water-in-oil (W/O) depending on the type of surfactant used.

In an O/W emulsion stabilized by an anionic surfactant, the O/W emulsion becomes a W/O emulsion at higher temperature in a phase inversion process. The temperature at which the phase inverts is known as the phase inversion temperature (PIT). At T<PIT, the O/W droplets are stabilized in solution by the surfactant. Once T=PIT, the surfactant becomes sufficiently hydrophobic that it does not necessarily stabilize one phase vs the other and the result is a bi-continuous phase within the initial oil droplet. At T>PIT, the surfactant becomes even more hydrophobic and would stabilize a W/O emulsion more readily. The droplet size of the resulting W/O emulsion is typically much less than the initial O/W emulsion because the phase inversion is constrained within each individual droplet.

A process for starch nanoparticle (SNP) synthesis using phase inversion is shown in FIG. 2. In brief, an oil phase is homogenized with a water phase containing fully cooked starch. Once the O/W emulsion forms, the temperature is raised to a temperature greater than the PIT for the particular surfactant and reaction conditions used. Once the phase inversion has completed, STMP is added and the reaction is allowed to proceed for 1 hour, forming internally crosslinked nanoparticles. An example of such a process is described in U.S. Pat. No. 6,755,915, which is incorporated by reference. Paraffin oil was used as the oil phase, with Tween 85 used as the surfactant. Tween 85 is an anionic surfactant with a critical micelle concentration (CMC) of 0.09 mM and a HLB of 11. As such, it is already relatively hydrophobic (compared to other hydrophilic stabilizers) but will initially form an O/W emulsion at low temperatures. To decrease the phase inversion temperature further, 0.3 M NaCl was used. Apart from this function, NaCl is a catalyst of the STMP crosslinking reaction as it screens electrostatic repulsion of the phosphates for nucleophilic attack. As reported by the patent, the phase inversion temperature of this emulsion is 25° C. Modifications were made to this procedure to optimize the amount of STMP needed to induce nanoparticle formation, leaving the parameters responsible for phase inversion (Tween 85 concentration, NaCl concentration, and volume fractions of oil and water phase) constant.

Examples

Cooking of Starch Granules.

Typically, 46.8 g of waxy maize starch was dispersed in 1 L of deionized water. To this mixture, 20.8 g of NaCl and 11.7 g of NaOH was added, and the whole was brought to 55° C. for 2 hours. The solution was allowed to cool and stored at 4° C. until ready for use.

PIE for STMP-SNP Synthesis.

In a typical reaction, 35 g of Tween 85 was dispersed in 600 mL of paraffin oil (oil phase) using a Silverson L4H high shear mixer stirring at 3000 RPM. Once the surfactant was fully dispersed, 400 mL of the cooked starch/NaCl solution in NaOH (water phase) was added and the shear rate was increased to 7500 RPM to form the emulsion. The temperature after complete homogenization of water and oil phase was ˜18° C. The shear force acting on the emulsion was enough to increase the temperature significantly, without any external heating required. At 55° C., STMP was added in solid form at various concentrations to initiate the crosslinking reaction. The amount of STMP added was varied with respect to the amount of starch in the reaction. More specifically, it was expressed as a mole percentage of the anhydroglucose units (AGU) of starch used in the reaction. Taking 5 mol % STMP as an example, there would be 5 STMP molecules for every 100 AGU in starch. To calculate the mass of STMP to be added the following formula was used

$m_{STMP} = {X \star \frac{n_{AGU}}{100\left( {1 - \frac{X}{100}} \right)} \star {305.885\frac{g}{mol}}}$

Where m_(STMP) was the mass of STMP to be added, X was the desired mol % STMP, n_(AGU) was the number of moles of AGU used and 305.885 g/mol is the molecular weight of STMP. Samples were synthesized using different values of X: 0, 1, 5, 10, 30 and 50 mol % STMP. The STMP would partition in the water phase as it was completely insoluble in the oil phase. The reaction was then allowed to proceed for 1 hour at 70° C.

To stop the reaction, 3 g of 37% HCl was diluted in 200 mL water and added to the emulsion. This neutralized the basic water phase so that nucleophilic attack of the starch hydroxyl groups to the STMP would be minimized and the crosslinking reaction stopped. The temperature was then brought down to 20° C. so that the continuous phase is aqueous (reversion to O/W emulsion). The particles were then precipitated using ethanol. To remove surfactant, the precipitate was washed three times with absolute ethanol and filtered using Buchner filtration. The nanoparticles were then dispersed in water and placed in a separatory funnel and allowed to stand so that oil could partition to the top of the dispersion. The water phase was then collected and any remaining oil was discarded. This was repeated 3 times to ensure any oil was removed. Finally, the sample was placed inside 10 kDa MW cut-off membrane for dialysis in a 1:10 ratio to dialysate to remove phosphate and chloride salts. Initially, the sample was dialyzed against 10 mM NaCl dialysate to lower the concentration gradient of salt from inside the membrane. This would prevent too much water from entering the membrane and rupturing it. It was then lowered by increments of half until day 4, when no salt was added. Dialysis was continued for 6 days, with initial dialysate changes occurring every 3 hours. After day 3, the dialysate was changed twice per day. Once dialysis was complete, the sample was frozen and stored at −20° C. for lyophilisation.

Dynamic Light Scattering and ζ-Potential.

In a typical measurement, 1 mg STMP-SNPs were dispersed in 1 mL milli-Q water (final concentration: 1 mg/mL) and placed in a low-volume disposable sizing cuvette for measurement in a Malvern Zetasizer (Nano series). For ζ-Potential, 1 mg of STMP-SNPs were dispersed in 1 mL 50 mM HEPES buffer (pH 7.6) and placed in a disposable zeta-cell for measurement. All measurements were performed at 25° C.

TEM.

The sample was prepared by dispersing 1 mg STMP in 1 mL water (final concentration=1 mg/mL) before placing 15 μL on a holey carbon grid and allowed to dry overnight. The next day, the samples were imaged using a Phillips CM-10 electron microscope.

ESEM.

A small amount of freeze-dried powder from the 0 mol % and 10 mol % samples were placed on a SEM sample holder with carbon tape. Compressed air was blown on the sample to remove loosely bound powder so that optimal imaging could be performed. The imaging was performed on a FEI Quanta Feg 250 ESEM.

P NMR.

STMPs were dispersed in milli-Q water at a concentration of 15 mg/mL and place in an NMR-tube. The proton-decoupled measurement was performed in an Avance 500 NMR spectrometer operating at 500 MHz (¹H) and 202 MHz (³¹P) using phosphoric acid as a reference and without a solvent lock. All samples were run with a delay of 5 seconds, a pulse width of 2.8 seconds and a sweep width of 398.35 ppm.

Water Retention Studies.

STMP-SNP samples prepared with different mol % STMP were dispersed in 1 mL water at a concentration of 15 mg/mL in a micro-centrifuge tube. These tubes were pre-weighed before the solution was placed in the tube. The samples were then centrifuged at 15000 RPM for 20 minutes. After centrifugation, supernatant was removed and the tube was re-weighed with the swollen product. The mass of the tube recorded before the experiment, as well as the mass of SNP present (15 mg) was subtracted from the final recorded weight to find the amount of water retained. For studies with salt, the sample which yielded the highest water retention (30 mol % STMP) was dispersed at a concentration of 15 mg/mL in 1 mL water in various pre-weighed tubes. Two salts (NaCl and MgCl₂) were then added at increasing concentrations. The samples were then centrifuged, the supernatant decanted, and weighed.

During the synthesis, several characterization techniques were performed to ensure that the intended processes were occurring. These included polarized light microscopy to ensure cooking of starch, optical microscopy to determine droplet size and conductivity measurements to determine the phase inversion temperature of the emulsion. In addition, several characterization methods were employed to determine the physical and chemical properties of the prepared STMP-SNPs. These include: dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), ³¹P nuclear magnetic resonance (NMR) spectroscopy, ζ-potential, and water retention studies.

First, complete cooking of starch needed to be confirmed before the PIE procedure could be initiated. If there were any bulk starch granules left in the water phase, crosslinking would occur from the granule, resulting in a significant increase in particle size. The complete dissolution of starch was confirmed by light microscopy with and without polarizers. If crystalline domains (such as those found in starch granules) were present, they would interact with polarized light giving rise to contrast from the background. No evidence of starch granules were seen after 2 hours of cooking as there appeared to be no granules left to interact with the polarized light.

To characterize the emulsion process, two methods were employed: conductivity and light microscopy. The former was to confirm phase inversion from O/W to W/O and the latter was to determine the droplet size. Before phase inversion, water would be the continuous phase and as such, any conductivity measurement would yield a high value. This is especially true since there were NaCl and NaOH present, which facilitates current flow. On the other hand, after phase inversion, oil would be the continuous phase. Since there were no ions present in the oil phase, the conductivity would drop significantly upon phase inversion. Using this principle, the conductivity was measured as a function of temperature, as shown FIG. 3. Before ˜30° C., the conductivity was high (˜11 mS/cm) as water was the continuous phase. Beyond this temperature, there was a rapid drop in conductivity to ˜100 pS/cm, correlating to the phase inversion of the emulsion. In addition, consistency of the emulsion changed significantly after phase inversion; below the PIT, the emulsion appeared thick and viscous whereas beyond the PIT, it appeared much thinner.

The droplet size was characterized before (A) and after (B) phase inversion using optical microscopy. Before phase inversion, droplet sizes were between 2-8 μm. After phase inversion, it was impossible to determine the droplet size as they were beyond the limits of the optical microscope resolution.

DLS and ζ-Potential

Dynamic light scattering (DLS) is used to measure the size of particles in solution. In principle, a sample is illuminated by a laser light and the amount of scattering is detected. For a very small time period after an initial measurement, the amount of scattering will be the exact same as the time period before it. It could therefore be said that at that time period, there is a full correlation between the two measurements. However, as time increases, this correlation will decrease due to the movement of particles by Brownian motion. Assuming full correlation corresponds to “1” and no correlation corresponds to “0”, a correlation function could be generated, which is normally in the form of an exponential decay. Smaller particles move very quickly, and therefore the correlation function would decay to zero at a short time period after measurement begins. Larger particles move much more slowly and therefore the correlation at higher time periods would decay to zero at longer times after the beginning of the measurement. In an ideal case, the correlation function can be modeled by an exponential function, where a relaxation time could be obtained for a particular species in solution. This relaxation time is related to the diffusion coefficient of the particulate species in solution. Using the Stokes-Einstein equation, and known parameters like temperature and viscosity, the hydrodynamic radius could be obtained. This is done automatically by the instrument software and what is obtained is an intensity plot that is generated from the correlation function which provides an idea of the population of particle sizes in solution. The peak of the intensity plot, or Z-average, is taken as the average particle size, which may be referred to as the particle size herein.

In addition to DLS, ζ-potential measurements were also performed on the STMP-SNPs prepared. If a charged particle is in solution, then it will attract counter ions to its surface via coulombic interactions. This layer of counter ions is called the Stern layer. The concentration of these counter ions decays with distance away from the particle and, this region is known as the diffuse layer. Collectively, the Stern layer and the diffuse region are part of the electrical double layer. At a certain point, there is no excess of one ion over the other, which corresponds to the bulk liquid phase. However, some of these solvated counter ions move with the particle itself and are considered to be “attached” to the particle. The point in the electrical double layer where this “attachment” stops is known as the slipping plane. In principle, the ζ-potential measures the difference in electric potential between bulk liquid and the slipping plane. From this, information about the particle surface charge can be obtained.

The intensity distribution of hydrodynamic diameter measured from DLS for samples prepared using different amounts of STMP is shown in FIG. 4 A. Without STMP, the free starch chains appeared to have a hydrodynamic diameter between 30-40 nm. Normally, a high MW polymer such as starch would have a larger hydrodynamic radius in solution. However, exposure to shear forces used in the process would have likely reduced the size of the native starch chains (resulting in a lower hydrodynamic radius), though the chains likely still have a MW of at least 100,000. When STMP was used, this diameter increased to between 300-400 at 30 and 50 mol %. In between these two extremes, there appeared to be some progression in the particle size from smaller to larger. This increase was likely due to the crosslinking of the starch chains within the emulsion droplets, resulting in a particle limited by the size of the droplets. Based on the sizes obtained at high STMP concentrations, it appeared that these droplets (and as such, the particles sizes) were between 200-400 nm. At lower concentrations, it was possible that less crosslinking was occurring and this limited the particle size to less than the droplet size. Particles of 300 nm or less, or alternatively up to 500 nm, could be obtained by reducing the particle size by increasing shear or altering the oil or surfactant.

The ζ-Potential measurements for each sample in pH 7.6 50 mM HEPES buffer is shown in FIG. 4 B. There was a general increase in the negative surface charge of the particles with STMP concentration. Even at 1 mol % STMP, there was a great deal of negative charge imparted on the particles. Quite likely, there was some phosphorylation at lower STMP concentrations but the nature of these species was dominated by phosphates that were not necessarily the phosphodiester linkage between separate starch chains (such as monophosphates).

Transmission Electron Microscopy (TEM)

TEM is a widely-used technique to visualize nanoparticles. Samples are loaded at low concentration (so that there is a very thin layer of material) on to a conductive grid and placed into a vacuum chamber. Electrons are fired at the sample at very high energy (100-1000 kV), either from a thermionic (where heat is used to release electrons) or a field effect emission (using a strong electric field). Electrons which interact with the sample are scattered, while others are transmitted through to a detector and an image is generated. TEM works best with electron-dense samples, such as metal nanoparticles, as the scattering would be much more obvious and detectable. Wth polymeric samples, such as starch, it is more complicated. Polymer chains themselves would not be able to be visualized since electrons may pass straight through them, without any reasonable contrast to the background. In addition, the high energy electrons would damage the polymeric sample quite easily. This being said, there are reports of crosslinked starch nanoparticles being imaged by TEM, likely due to the increase in electron density.

TEM was performed on the STMP-SNPs at various concentrations of STMP and typical images are shown in FIG. 5 A-F. Without STMP, small spherical particles of ca. 20 nm were seen. These may be due to dried appearance of the free starch chains, small droplets of oil remaining from the purification process, or simply an artifact of drying itself. At 1 mol % STMP, faint areas of darker, fibrous features indicated that there was indeed an effect on the particle morphology even with a small amount of STMP. Clear particles were seen beyond 5 mol % STMP where darker areas correspond to the dense, internally-crosslinked core of the nanoparticle, with lighter representing sparser crosslinking on the outer regions. These particles were not strictly uniform in shape but were confined between 100-700 nm in size, with isolated larger (>1 μm) and smaller (<100 nm) particles. In addition, individual chains in the crosslinked polymer network could be seen at higher concentrations of STMP. Based on this, it was more accurate to refer to this material as a nanogel (i.e. hydrogel nanoparticle) as opposed to a solid particle. Since these nanogels were dried out for the TEM experiment, it may not necessarily reflect the solution morphology. This being said, the previously-obtained DLS data supports the TEM imaging, suggesting that the solution behaviour was not far from the dried morphology. A hydrogel may “swell” meaning that it takes in water from a solution, and de-swell meaning that it releases water when dried or under other conditions, but the outer diameter of the particle does not necessarily vary in proportion to the swelling.

Environmental Scanning Electron Microscopy (ESEM)

SEM is another imaging technique that uses electrons to visualize samples at a much higher resolution than light. Unlike TEM, however, SEM relies on electrons that are scattered out of the sample upon bombardment rather than the electrons that are transmitted through the grid for contrast. The basic principle is that some electrons on the sample surface are ejected from the sample upon impact with the primary electron. These electrons (called secondary electrons) are then collected by an electric field where they hit a phosphor screen, emitting flashes of light. This light is then amplified using a photomultiplier tube and ultimately detected using digital electronics. The resulting 2-D image is a collection of intensities corresponding to the angle of incidence of the electrons on the surface of the sample. High incidence angles result in more electrons being emitted, causing steeper morphologies to appear brighter. Typically, this is done on conducting samples such as metals since the electrons can flow through the material freely without building up at the surface. If non-conducting samples (such as SNPs), significant charge build-up at the surface causes charging effects that significantly lowers the quality of the image obtained.

A variation of this technique is environmental SEM (or ESEM), where chamber pressures are kept relatively high and water molecules are abundant in the sample chamber. As a result, charging artifacts are removed even in non-conducting samples and the image quality is improved. Sample preparation for ESEM is unchanged from conventional SEM. ESEM was performed on the 0 mol % (A) and the 10 mol % (B) STMP-SNP samples as shown in FIG. 6. Without STMP, no obvious morphologies or particle formation was observed. However, with 10 mol % STMP, spherical particles between 300-500 nm were observed, consistent with both DLS and TEM obtained previously. It was important to note that the sample was lyophilized prior to imaging with ESEM, unlike TEM where the sample was dried in air on the grid. This meant that the morphology obtained from ESEM would more closely reflect that in solution, and there would be no drying effects. Therefore, in solution, it was likely that the particles were uniformly spherical.

P-NMR Spectroscopy

To gain an idea of the nature of the phosphate species present in the samples prepared, P NMR spectroscopy was performed. Certain atomic nuclei, when placed in a magnetic field, can absorb specific wavelengths of light in the electromagnetic spectrum. This is due to the fact that these nuclei have special spin states which will either align with or against an external magnetic field. For example, with a spin % nuclei, two spin states are present; one will align with the magnetic field and the other will oppose it. This generates an energy gap, the size of which is dependent on the specific nuclei present, and its local electronic environment. The spin state of lower energy could be excited to a state of higher energy if electromagnetic radiation of a frequency corresponding to the characteristic energy gap was applied. This absorption of radiation by specific spin states in the external magnetic field forms the basis of NMR spectroscopy. As mentioned previously, the local electronic environment plays a major role in this energy gap. This is because electrons can also align themselves in the magnetic field to generate their own, weaker, magnetic field which opposes the external, stronger, magnetic field. Effectively, this shields the nuclei being probed from the external magnetic field, resulting in a different absorption radiation frequency. These changes are quite small, with shifts on the order of Hz over a MHz reference signal. As a result, the ratio of the change to the reference is on the order of a 10⁻⁶. Therefore, these values are typically multiplied by 10⁶ before analysis. This modified ratio is known as the chemical shift (δ) and is normally in the units of parts-per-million (ppm). The chemical shift can also be a negative number, as it is measured as a change relative to a reference frequency.

Phosphorus (P), unlike many other nuclei studied using this technique, has a spin ½ nuclei of 100% abundance, meaning that all phosphorus atoms could be probed using this technique. This being said, P-NMR, in general, is not quantitative; uneven nuclear-Overhauser effect (NOE) enhancement prevents any integration of the peaks.¹¹⁸ However, the chemical shifts can provide evidence of crosslinking within the STMP-SNPs, and the other types of phosphate species present. In addition, the sharpness of the peaks would indicate whether there is covalent attachment to the polymer chain (broadened peaks) or whether inorganic phosphate species are simply embedded in the crosslinked nanogel non-covalently (sharper peaks). This reaction has been studied with starch, as mentioned previously. The data obtained from NMR was compared to literature reports consistent with the chemical shifts observed for assignment of the peaks.

The P-NMR spectra for all samples prepared are shown in FIG. 7 A-F. As expected, no organic/inorganic phosphorus species were detected for the sample prepared without STMP (FIG. 7 A). At 1 mol % STMP (FIG. 7 B), very small and poorly resolved peaks appeared at 0.56 and 3.53 ppm, potentially consistent with phosphodiester and monophosphate respectively. There was also the appearance of the outer phosphorus atoms of the starch triphosphate at −7.65 ppm. There should be a corresponding peak for the inner phosphorus of the triphosphate at ca. −20 ppm. However, due to the poor resolution obtained for this sample, this was not assigned. At 5 mol % STMP (FIG. 7 C), multiple new peaks appeared indicative of more extensive phosphorylation. Between 0 and 5 ppm, there were 3 peaks. Based on the TEM image, it was known that there was some degree of crosslinking. However, it was not clear which of these peaks corresponded to the phosphodiester or monophosphate. In addition, the sharp peak at 1.33 ppm seemed to indicate an inorganic species, potentially inorganic monophosphate that was not removed by dialysis. In addition, 3 peaks were seen between −5 and −10 ppm. One of the peaks may correspond to the α and γ phosphorus atoms of the triphosphate, another may be the same but for inorganic triphosphate and the last one may be for the presence of the diphosphate. Finally, the last peak at −21.8 ppm was likely to be the β phosphorus of the either the organic or inorganic triphosphate. For 10 mol % STMP (FIG. 7 D), there were fewer peaks, and those that were present were quite broad. This was strongly indicative of only organophosphate species being present as opposed to inorganic phosphates. Likely, dialysis was very successful on this sample and as such, a cleaner spectrum was obtained. This being said, the two peaks between 0 and 5 ppm were likely to be starch monophosphate or phosphodiester linkages, but it was not possible to distinguish between them. For 30 mol % and 50 mol % (FIGS. 7 E and F, respectively), there was even more significant broadening of the peaks between 0 and 5 ppm, likely indicating significant crosslinking of starch. In addition, several other peaks were seen, including the outer phosphorus atoms of the organic triphosphates and diphosphates between −5 and −10 ppm, with sharper peaks in this region corresponding to the inorganic analogues to these species. At these high concentrations of STMP, it was quite likely that even 6 days of dialysis was not enough to remove the high amount of inorganic biproducts. Another complication could be that the crosslinking was so extensive, that it trapped unreacted and inorganic species within the crosslinked nanogel, preventing escape through dialysis processes.

While the locations of the peaks generally corresponded to expectations (e.g. triphosphates vs monophosphates), absolute assignments proved difficult due to the wide range of possibilities and the small differences in chemical shift between certain organophosphate species (e.g. phosphodiesters vs monophosphates). Furthermore, chemical shifts may differ depending on which hydroxyl group in the sugar ring the phosphate species was bound to. To resolve these peaks definitively, it may be necessary to be break down the STMP-SNPs into smaller macromolecules by using enzymes (such as amylase) and performing P-NMR again. This would sharpen each individual peaks to a point where assignment could be possible.

Water Retention of Nanogels

One interesting property of the prepared nanogels is their ability to retain water and swell, much like a macroscopic gel. In typical crosslinked nanogels, the degree of swelling is inversely proportional to the degree of crosslinking; a higher crosslinking density prevents the polymer chains from expanding in water. A plot of STMP concentration vs mass of water retained, as well as an iodine stain of the swollen gel is shown in FIGS. 8 A) and B) respectively. Surprisingly, a significant amount of water was retained at very high STMP concentration. Despite TEM images clearly showing crosslinked particles at lower concentrations of STMP, less water retention was observed. A possible explanation for this could lie in the inefficiency of the crosslinking reaction itself. As evident from NMR, apart from peaks attributed to crosslinking, there is a much higher degree of triphosphates present. The presence of charged groups within a crosslinked network can contribute to swelling through electrostatic repulsion. At higher concentrations of STMP, the increase in organic triphosphates could induce significant electrostatic repulsion within the STMP-SNPs, leading to a higher degree of swelling. However, these triphosphates were not as present at low STMP concentrations (relative to monophosphates and phosphodiesters). As a result, there is less swelling at lower concentrations. Drug loading may be related to swelling and so STMP concentration in the range of 20-50 mol % (which is calculated herein based on starch AGU), or 20-40 mol %, or 30-50 mol %, or about 30 mol %, may be preferred for nanoparticles used to deliver a drug.

To further investigate the nature of swelling of the nanogels, the effect of salt on the water retention of the 30 mol % STMP-SNP sample was tested. Salts can screen the electrostatic repulsion within the polymer network, and potentially decrease swelling. The water retention as a function of salt concentration, as well as an iodine-stained image of the centrifuged 30 mol % STMP-SNPs as a function of NaCl concentration is shown in FIGS. 9 A) and B) respectively. For both salts, there was a significant decrease in the amount of water retained by the gel. This decrease was more gradual with NaCl compared to MgCl₂, likely reflecting the fact that Mg²⁺ is a divalent ion and would more effectively screen the electrostatic repulsion than Na⁺. Interestingly, the swelling was not completely prevented; about 0.2 g of water was still retained at high concentrations of both salts. It would be unlikely for complete dehydration of the gel to occur, especially to ensure that the ions stay within the polymeric nanogel.

Conclusion Re: Particle Synthesis and Characterization

The PIE process to make STMP-SNPs was successful based on the several characterization techniques performed. Firstly, the synthetic concept of phase inversion was confirmed from conductivity measurements, which showed a decrease in conductivity, and a corresponding decrease in droplet size. Once particles were purified and dried, the particle size and morphology varied with the amount of STMP used in the synthesis, with clearly-defined internally-crosslinked nanoparticles present at least at concentrations of 5 mol % and more. As a result, it was more accurate to call the STMP-SNPs “nanogels”, as opposed to a solid (i.e. densely packed) particle. In general, the particle size and negative charge increased with increasing amounts of STMP, as evident from DLS and ζ-potential. From TEM, the crosslinked chains were clearly visible within the densely-crosslinked core. The phosphorylation of the samples was confirmed by P NMR, showing various species present after purification. This being said, individual peaks were not assigned as there were many potential possibilities. Therefore, further P NMR work needs to be done (such as spiking experiments and enzymatic digestion) to resolve these peaks and definitely assign them. Finally, the nanogels displayed swelling behaviour; a significant amount of water was retained upon dissolution. In general, the water retained was lower when <10 mol % STMP was used. It was highest at 30 mol % STMP, with 50 mol % being slightly lower. This was potentially due, in part, to influence from very charged phosphate groups, causing the internal phosphates to repel each other, leading to increased swelling. These large phosphate groups were present in greater amounts at higher STMP concentration. Increasing salt concentration also resulted in a lower amount of water retained likely due to screening of this electrostatic repulsion. While there was extensive characterization performed, more techniques (such as viscosity measurements) will need to be done in the future to further understand the nature of the nanogels.

Drug Delivery

After successful preparation and characterization of phosphate crosslinked nanoparticles, the potential application in drug delivery was explored. For this purpose, confirmation was obtained that the nanoparticles were biocompatible (not toxic to cells). This was done using an MTT assay. An MTT assay is normally used to determine in-vitro cell viability/toxicity. The MTT reagent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium, is internalized by live cells and converted to a formazan by oxidoreductase enzymes, which has a strong purple colour. This purple colour could be quantified by absorbance at 570 nm. A strong purple colour indicates no cell toxicity, while a non-existent purple colour indicates high toxicity. In addition, another aspect of drug delivery is the ability of the nanoparticle to load and release drug. Since the linkage formed between the starch chains is negatively-charged (due to the phosphate group), there was an inherent limitation on the types of drugs that could be loaded within the crosslinked starched network. For example, hydrophobic drugs like docetaxel were unlikely to partition into the porous structure and instead precipitate out as free docetaxel. However, a positively charged drug (such as doxorubicin, with pK_(a)=9.53), would preferentially partition into these pores and load effectively. In addition, doxorubicin (DOX) is fluorescent (λ_(excitation)=490 nm, λ_(emission)=590 nm), and quantification of the release could be done using fluorescence-based assays.

Examples

MTT Assay.

Typically, HeLa cells were seeded into 60 wells of a 96 well plate at a concentration of 5000 cells/well and left to proliferate (grow and divide) overnight. The next day, the sample was washed with phosphate-buffered saline (PBS) and 100 μL of cell medium was added. At this time, 100 μL of 10 mg/mL 0 mol % STMP and 30 mol % STMP samples were added to the first well (final concentration 5 mg/mL) and a serial dilution was performed so that the next well was half the concentration of the previous well. In other words, the most concentrated well was 5 mg/mL, followed by the next well which was 2.5 mg/mL, and this dilution continued until well 9. Well 10 was reserved as a control for no sample. Since the assay was run in triplicates, each sample would be allocated 30 wells (10 for each series with 2 duplicates). The cells were left to incubate with the sample overnight. The next day, 25 μL of 5 mg/mL MTT reagent was added and allowed to be internalized into the cells for 2 hours. The cells were then lysed (broken apart) by pH 4.7 dimethyl sulfoxide (DMSO) to release and dissolve the purple formazan created in the cells. After 4 hours of incubation, the absorbance at 570 nm was measured using a SpectraMax M3 spectrometer. The cell viability was calculated according the following equation:

${{Cell}\mspace{14mu} {Viability}\mspace{14mu} (\%)} = {\frac{A_{sample}}{A_{control}} \star {100\%}}$

Where A_(sample) was the measured absorbance of the sample well and A_(control) was the measured absorbance of the control well (without any sample).

DOX Calibration Curve.

From a stock DOX solution of 1 mg/mL in water, several dilutions were made so that a final concentration of 5 μg/mL was reached. The fluorescence of this solution was measured using a Varian spectrometer using an excitation wavelength of 490 nm and observing the emission peak at 590 nm. A calibration curve was generated using 5 μg/mL as the highest concentration to 0.01 μg/mL as the lowest. A fit of the plot was found using linear interpolation constraining the intercept to 0.

Drug Loading.

In a typical loading experiment, 100 μg of DOX was mixed 100 μg of STMP-SNPs in 1 mL of 50 mM buffer and incubated for 4 hours. After incubation, the samples were centrifuged to separate bound drug from loaded drug. The fluorescence of the supernatant at 590 nm was measured using fluorescence spectroscopy using an excitation wavelength of 490 nm. The drug loading capacity was then calculated according to the following equation

${{Loading}\mspace{14mu} {Capacity}\mspace{14mu} (\%)} = {\frac{D - \frac{F}{S}}{D} \star {100\%}}$

Where D was the total drug added (in all cases 100 μg), F was the fluorescence of the free drug measured after centrifugation, and S was the slope of the calibration curve within the range of detection (12.454 a.u./(μg/mL)).

Drug Release.

For drug release, STMP-SNPs were loaded at the optimal conditions determined from previous experiments, dispersed in 1 mL of 50 mM buffer, and placed within a 3500 Da molecular weight cut-off dialysis membrane. The sample was then dialyzed against 19 mL of a 50 mM buffer (Total volume=20 mL). The fluorescence of the dialysate was measured using fluorescence spectroscopy. The released drug was calculated according to the following equation:

${{Release}\mspace{14mu} (\%)} = {\frac{F}{L \star S} \star X \star {100\%}}$

Where F was the fluorescence intensity of the dialysate (a.u.), L was the loaded drug concentration (μg/mL), S was the slope (12.454 a.u./(μg/mL)), and X was the dilution factor due to the volume of dialysate (20).

Drug Release in Culture Environments.

The 30 mol % STMP-SNPs (100 μg) were loaded with DOX to capacity in 1 mL 50 mM pH 7.6 HEPES buffer, washed three times with water and finally redispersed in 1 mL PBS, DMEM, 10% FBS and 100% FBS, before leaving to mix. The samples were centrifuged at the required time point and 10 μL of the supernatant was diluted to 590 μL of PBS for measurement in a Varian fluorescence spectrometer. Using the calibration curve, released DOX was calculated using the following equation:

${{DOX}\mspace{14mu} {Released}\mspace{14mu} (\%)} = \frac{F}{S \star L}$

Where F was the fluorescence of the supernatant measured by spectroscopy (a.u.), S was the slope of the calibration curve (a.u./(μg/mL) and L was the loaded drug concentration (for 30 mol % STMP-SNP: 40 μg/mL).

MTT Assay

As mentioned previously, the MTT assay was used to determine if the synthesized particles were toxic by themselves to the cancer cells. The cell viability plots for both the 0 mol % and 30 mol % STMP samples are shown in FIG. 10. Overall, there was very little toxicity seen in both samples. Though there was a noticeable decrease in the viability at 2.5 and 5 mg/mL for the 30 mol % sample, this was also seen in the 0 mol % sample. This suggested that either starch itself was toxic beyond 2.5 mg/mL (though this was unlikely due to the fact that this was studied with other SNPs previously) or there may have been some chemicals (oil or surfactant) which may not have been removed during the purification process. Another potential reason was the dilution of the cell medium at very high concentrations. Due to use of serial dilutions, the well containing the highest concentration of sample would have the lowest concentration of medium. This could have affected the rate of cell proliferation. In any case, this effect was not very pronounced and only small decreases in the cell viability were noted. Furthermore, a concentration of 2.5 mg/mL is already quite high for clinical applications. While many data points showed that there seemed to be increased cell proliferation at low concentration of sample, the wide error bars suggested that this effect may be due to the natural variation of cell proliferation in a specific well.

Drug Loading and Release

Before any drug loading experiments were performed with prepared STMP-SNPs, a calibration curve for free DOX was generated as shown in FIG. 11, showing the fluorescence intensity as a function of DOX concentration. In addition to being fluorescent, DOX is also quite coloured; at higher concentrations, inner filter effects affected the linearity of the calibration curve. As such, a linear fit could only be found from 0 μg/mL to 5 μg/mL.

DOX is water-soluble, implying that if there is no loading, the drug would remain in the supernatant after centrifugation while the nanoparticles settle to the bottom. The drug loading for STMP-SNP samples prepared with 30 mol % STMP was determined at different pH as shown in FIG. 12 A. The buffers used were: 50 mM HEPES (pH 7.6), 50 mM citrate (pH 6), 50 mM acetate (pH 4). Drug loading was nearly 4-fold higher at pH 7.6 compared to pH 4 or 6. This was likely due to the fact that there were more phosphate groups deprotonated at higher pH, giving the STMP-SNPs a more negative charge. The pk_(a) of DOX (9.53) ensures that the drug is also quite positively charged. This means that electrostatic interactions are stronger between the DOX and the STMP-SNPs at higher pH, resulting in a higher loading. At lower pH, drug loading is limited as most phosphate groups would be protonated with the exception of the phosphodiester and monophosphate species. It can be concluded, therefore, that the presence of larger phosphate species (such as diphosphates and triphosphates) provides significant contributions to the drug loading. At pH 7.6, the loading capacity achieved was ˜40%. This meant that for 100 μg of SNP, 40 μg of DOX was bound. Compared to previously-studied smaller SNPs (about 50-150 nm, no-crosslinker) where the loading capacity was 0.05%, this represented a ˜800-fold improvement in loading capacity. This could be attributed to the larger size of the nanoparticles, as well as the introduction of highly negatively charged phosphate groups which aid in the drug binding.

Once the optimal drug loading pH was known, drug was loaded on to the starch nanoparticles at pH 7.6, followed by release studies using dialysis at different pH. The release profiles for this experiment are shown in FIG. 12 B. Within the first 1-2 days for all three samples, there is a sharp release of drug from the nanoparticle which could be due to drug that is more loosely bound or due to the sharp concentration gradient between the dialysate and the sample. Beyond this, the released drug increases slowly with time reflecting the diffusion of the more tightly-bound drug. The amount of drug released was significantly greater at lower pH compared to that the loading pH. This could also be explained by the weaker electrostatic interactions between the drug and the STMP-SNP, as discussed previously. This release at lower pH was extremely desirable for drug delivery applications, as cancer cell interiors tend to be more acidic than physiological pH. Therefore, internalization of the loaded STMP-SNPs would lead to a significant “burst” release in cells.

Next, the effect of STMP concentration used in the synthesis on the drug loading and release profiles was investigated. Evident from NMR, there is a higher amount of triphosphates at 30 and 50 mol % STMP relative to monophosphates compared to lower concentrations where monophosphates and phosphodiester linkages dominate. The drug loading capacities in 50 mM HEPES buffer at pH 7.6 for the various STMP-SNPs are shown in FIG. 13 A. In general, the loading capacity increases with STMP content until 30 mol %, with a slight decrease thereafter. The increase in drug loading capacity is attributed to a higher concentration of phosphate species (especially di- and tri-phosphates) within the crosslinked nanoparticle. However, the decrease with the 50 mol % sample went against the trend. Since there were more available phosphates, there should have been a higher drug loading capacity if the drug loading was purely electrostatic in nature. An interesting comparison for this drug loading data was the swelling studies previously performed. It was seen that with 50 mol % STMP, there was slightly less water retained in the nanogel compared to 30 mol %. It was quite possible that, in addition to electrostatic interactions, there was also significant influence from the volume of the nanoparticle to “store” the drug on the overall drug loading. It would also explain why there is a large jump in the capacity between 10 and 30 mol % STMP. However, it is expected that the capacity increases somewhere between 10 and 30 mol % STMP, for example 20 mol % STMP.

The release of the loaded STMP-SNPs at different STMP concentrations was monitored at pH 4 and the profile is shown in FIG. 13 B. The sample prepared without STMP showed a complete burst release, implying that the drug was only loosely bound to this material. All samples prepared with STMP had a much more gradual release, indicating stronger drug/SNP interactions. There was no obvious trend in the release profile with STMP concentration. A minimum of drug was released (relative to the loaded drug) at 10 mol % STMP as compared to 30 or 50 mol %, where a higher percentage was released. One possible explanation is the protonation of the triphosphates and diphosphates at low pH. From P-NMR, it was evident that there was an abundance of larger organic phosphates which contributed to the high loading, as discussed previously. However, in terms of release, the protonation of these groups created a large concentration gradient of free drug from inside and outside the dialysis membrane, resulting in more drug released. At low STMP concentrations, there likely were more monophosphates/phosphodiester linkages (relative to larger phosphate species) which bound less DOX, but were not deprotonated at low pH. Therefore, a lower percentage of DOX was released.

Finally, to investigate the effect of cell culture environment on the drug release, the loaded STMP-SNPs were placed in various environments. These included phosphate-buffered saline (PBS), Dulbecco's Modified Eagle Medium (DMEM), 10% Fetal Bovine Serum (FBS) in DMEM, and 100% FBS. PBS is typically used as a sodium phosphate buffer formulation containing various salts to match physiological ion concentration, as well as pH found in blood. DMEM is the typical medium used for culturing many cell lines (including cancer cells) and contains vitamins, glucose and essential amino acids for cell growth and proliferation. FBS is the supernatant of centrifuged blood of a bovine fetus after adding a coagulant. This normally contains several proteins typically found in human blood, a major component of which is bovine serum albumin (BSA).

The DOX release in these environments at 2 and 36 hours after redispersion are shown in FIG. 14. Even for PBS, significant desorption of drug was seen after just 2 hours, even though the pH of this buffer was 7.4. This could be attributed to the high salt concentration (>100 mM) which may have screened the attractive electrostatic interactions between the SNPs and the drug. In addition, the presence of the phosphates in the buffer may have attracted bound drug from the SNPs to the free solution. There was no major difference in the released drug after 2 hours across all the environments studied with ˜30% of the drug released on average. After 36 hours, the sample incubated in 100% FBS showed complete desorption of DOX, whereas PBS had released ˜60% of the loaded DOX. Many of the proteins in the FBS likely contain positively charged residues (e.g. lysine or arginine) which could effectively displace the doxorubicin from the SNPs. However, this likely required more time due to the proteins being more bulky than the salts in PBS and subsequently, steric hindrance slowed the adsorption of the proteins to the SNPs.

In an ideal case, the drug would be adsorbed within the network of the STMP-SNPs such that larger macromolecules (such as proteins) would not be able to penetrate and displace it. However, it appeared that the pore size of the nanogel permitted these larger molecules to diffuse in, affecting the drug release. Another potential explanation was that most of the DOX was adsorbed only on the surface of the STMP-SNP (and not within the crosslinked network). Therefore, it would be much easier for the aforementioned molecules to disrupt the adsorption. However, the release of only about 30% of the drug over 2 hours is already suitable for use in the treatment of cancer.

Conclusion Re: Drug Delivery

In general, the prepared STMP-SNPs were non-toxic as seen from the MTT assay. Any small deviation from the control did not show any specific trend, implying that the differences were likely due to natural variation in cell proliferation rates. This being said, a small decrease in cell viability was seen in both the 0 mol % and 30 mol % STMP-SNPs. This was potentially due to residual impurities from the PIE process or dilution of the cell medium at higher sample concentrations.

Drug loading and release experiments conducted in simple environments (only buffer) showed significant promise. More specifically, loading capacity for the 30 mol % STMP-SNPs at pH 7.6 was 40%, representing an 800-fold improvement over SNPs previously studied. In addition, the release was accelerated at lower pH, which was more desirable as cancer cell environments are more acidic than physiological conditions. Generally, there was increase in drug loading with STMP concentration used to prepare the nanogels. This reaches a maximum at 30 mol % (˜40% loading), tapering off slightly with 50 mol % (˜30% loading). Curiously, this trend reflected the ability of the nanogels to retain water, with the highest drug loading occurring in the same sample which retained the water. This suggested that, in addition to electrostatic interactions, the swelling behaviour also influenced the drug loading within the STMP-SNPs. However, when loaded STMP-SNPs were placed in more typical cell culture environments, drug release was much quicker. It was likely that increased salt concentration, and the presence of proteins/other interferences caused the adsorbed drug to desorb from the nanoparticles. This suggests that the nanoparticles may be preferred for use in delivering drugs to mucous or aqueous environments, for example to the digestive system, mouth, nose, or throat.

Nanoparticles are made using a food-grade crosslinker, STMP, to increase the size of the nanoparticles over starch strands through the formation of covalent phosphate crosslinks. The challenge lay in confining the particle size so that this process was not completed in bulk. To do this, an emulsion based protocol was followed based on a patent held by the collaborating company. In brief, a phase inversion emulsion allowed for sufficiently small droplet size to be formed using low to moderate energy methods (such as high shear mixing).

The phosphate-crosslinked SNPS (STMP-SNPs) were successfully prepared using different amounts of STMP during the crosslinking process. They were characterized using TEM, DLS and SEM. In general, the particle size increased with increasing STMP concentration, with more obvious nanoparticles observed at STMP concentrations higher than 5 mol %. The STMP-SNPs were negatively charged due to phosphorylation, while the sample prepared without STMP was neutral, as measured from ζ-potential. The particle morphology, with interconnected regions of crosslinked chains suggested that these were nanogels, rather than solid particles. P NMR was performed on the STMP-SNPs and in general, many different organic phosphorus species, such as monophosphates and diphosphates, were present along with the phosphodiester linkages. However, enzymatic digestion of the STMP-SNPs may be necessary in order to definitively resolve the peaks obtained from the spectra as significant broadening was present. At high STMP concentrations, the prepared nanogels retained significant amounts of water. This was due, in part, to the electrostatic repulsion of the larger phosphate species within the crosslinked polymer network. Confirmation of this electrostatic repulsion mechanism was found from the addition of salt to the nanogels, which significantly reduced the amount of water retained.

The performance of the STMP-SNPs as a drug delivery vehicle was explored. Firstly, an MTT assay was used to determine any toxicity to the HeLa cancer cell line. It was found that the samples containing STMP did not show any significant toxicity as compared to the sample without STMP. For both samples, toxicity seemed to increase at 2.5 mg/mL but may likely have been due to either impurities from the emulsion process or medium dilution. The drug loading studies indicated that the model drug, DOX, was optimally adsorbed on to the STMP-SNPs at pH 7.6, and the loading capacity was 800-fold higher than previously-studied SNPs. The release of the drug from the STMP-SNPs was much quicker at lower pH, likely reflecting the protonation of the phosphate species in a more acidic environment. It was also seen that the STMP-SNPs prepared with 30 mol % STMP had the highest drug loading, which likely reflected the swelling behaviour of this particular sample.

One potential way to improve the crosslinking would be to add a divalent (or event trivalent) metal ion, in addition to the NaCl used in the study, as a catalyst during the PIE process.

With regards to drug delivery applications, the current study suggests that DOX-loaded STMP-SNPs are very susceptible to drug release under typical cell culture environments. This may be due to simple surface attachment of the DOX to the STMP-SNPs and could be solved by incorporating the DOX into the nanoparticles during the PIE process (i.e. by adding the DOX to the water phase in either emulsion or while the water phase is initially being prepared) to ensure that it would be internalized in the nanogel structure. 

We claim:
 1. A nanoparticle comprising starch strands crosslinked with a phosphate crosslinker.
 2. The nanoparticle of claim 1 having a peak intensity or Z-average size determined by DLS in the range of 80-500 nm.
 3. The nanoparticle of claim 1 with a chemotherapeutic drug loaded into the nanoparticle.
 4. The nanoparticle of claim 1 with a targeting ligand.
 5. The nanoparticle of claim 1 made with 20-50 mol % STMP.
 6. A process of treating a disease of the nose, mouth or throat comprising a step of applying a nanoparticle of claim 1 to the nose, mouth or throat, or of treating a cancer comprising a step of injecting nanoparticles according to claim 1 into blood of an animal, which may be a human.
 7. A process of making nanoparticles using a phase inversion emulsion process wherein starch is is cross-linked with a phosphate cross-linker.
 8. The process of claim 7 wherein a drug is included in the water phase.
 9. The process of claim 7 wherein the crosslinker is STMP. 